The invention relates to a process for the preparation of pilocarpine from suspension cultures or differentiated in vitro plant cultures of the genus Pilocarpus. The woody plant of the genus Pilocarpus with its most important representatives P. jaborandi and P. microphyllus, which is indigenous to South America, contains, inter alia, the imidazole alkaloid pilocarpine.
Pilocarpine is used as a pharmaceutical or therapeutic substance. It is a parasympathomimetic which acts directly. It has a muscarine- and acetylcholine-type action with the advantage that it cannot be degraded by choline esterase. It enhances the secretion of the perspiratory glands and salivary glands and stimulates the smooth muscles of the bowels and of the bronchial tubes. Nowadays, ophthalmology is virtually the only field of application. Since, as a myotic, it reduces the intraoccular pressure, it is preferably employed for treating glaucoma.
Obtaining pilocarpine via a chemical or biochemical route has proved to be difficult and, so far, uneconomical. The demand for pilocarpine is therefore substantially covered from the plant itself by isolation and purification of the alkaloid. However, the seeds of Pilocarpus retain the ability to germinate only for a very brief time, as is also the case in many other tropical plants. The germination percentage drops by more than 90% within a few weeks. This means that Pilocarpus seedlings can only be successfully grown near the sites in which they grow naturally. Another disadvantage is the slow growth of seedlings under greenhouse conditions.
On the other hand, attempts to culture Pilocarpus in vitro have failed so far. In general, the success of an in vitro culture of woody plants, in particular from callus, can only be ensured in exceptional cases, and this is only the case with relatively undemanding species which do not present problems (Bonga and Durzan (1987), Cell and Tissue Culture in Forestry, Vol. 1-3, Martinus Nijhoff Publishers; Chalupa (1987) in: Bonga and Durzan (1987), 1.c.; Wann (1988), Horticultural Reviews 10, 153). Besides the fact that in vitro culture as such is difficult, it is also extremely doubtful if the resulting in vitro cultures are still capable of synthesizing acceptable amounts of the desired natural product, in this case pilocarpine. A large number of such cultures have proved to be useless for practical purposes.
It was therefore the object to develop a process for the preparation of pilocarpine from plant material on the basis of an in vitro culture capable of alkaloid formation, so that the above-mentioned disadvantages as regards site and growth conditions can be avoided.